Identification and applications of porcine reproductive and respiratory syndrome virus host susceptibility factor(s) for improved swine breeding and development of a non-simian recombinant cell line for propagation of the virus and a target for a novel class of antiviral compounds

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses in swine. The present invention determined that CD 151 is a susceptibility factor for PRRSV infection by transfecting a cell line which is not susceptible to PRRSV infection (BHK-21) with CD 151, which rendered the cell line susceptible. Because CD 151 can be accessed in cellular material including blood platelets and germplasm, the present invention provides a non-invasive method of screening different swine for susceptibility to PRRSV, thereby improving breeding plans. In the case of a valuable animal, results from such screening can indicate any offspring&#39;s susceptibility to PPRSV. Additionally, the viral RNA-CD 151 interaction possesses high affinity and can be used as a tool to detect anti-viral compounds which can be further improved by using combinatorial chemistry. Accordingly, anti-viral compounds that can block the viral RNA-CD 151 interaction can be developed. Advantageously, transfection of CD 151 into non-simian cell lines can confer susceptibility to PRRSV and these recombinant cell lines can be used for preparation of biologics that will avoid simian cell lines which could be a source of primate viruses in xenotransplanted organs from pigs. Finally, the present invention describes the basic mechanism by which the virus RNA enters a target cell. This novel class of proteins is termed viral RNA entry proteins and a novel class of compounds named anti-RNA Entry Proteins can be used to block the entry of viral RNA, thereby preventing viral infections.

RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.09/772,044 filed Jan. 29, 2001 now U.S. Pat. No. 6,740,490.

SEQUENCE LISTING

A printed Sequence Listing accompanies this application, and has alsobeen submitted with identical contents in the form of acomputer-readable ASCII file on a floppy diskette and a CDROM.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed towards the screening of different pigbreeding lines by quantitative reverse-transcriptase polymerase chainreaction (RT-PCR) for CD 151 and/or DNA PCR using porcine CD 151.Selection of swine lines which are genetically low in or do not possessCD 151 produces offspring which are less susceptible or not susceptibleat all to infection by porcine reproductive and respiratory syndromevirus (PRRSV). The present invention also concerns non-invasive methodsfor screening live pigs and swine germplasm for susceptibility to PRRSV.Additionally, the invention describes the development of cell lines forpropagating high titer stocks for making killed and modified live virusvaccines in non-simian cell lines. Moreover, the invention describes anovel plasmid, useful in transforming previously non-susceptible celllines into susceptible ones for producing the titer stocks used invaccines. The invention also pertains to providing a tool for discoveryof a novel class of anti-viral compounds that will block the interactionbetween CD 151 and PRRSV 3′-UTR RNA. This novel class of compounds hasbeen termed anti-RNA Entry Proteins (anti-REPs).

2. Description of the Prior Art

Porcine reproductive and respiratory syndrome (PRRS) is a RNA viruswhich emerged in the late 1980's as an important viral disease of swine.PRRS is the most important, economically significant disease of theswine industry in the United States and around the world. The origin andevolution of PRRSV is not known. Thus, it is a novel emerging virus ofpigs. This disease, which has previously been referred to as “mysteryswine disease”, “swine infertility and respiratory syndrome”, or “blueear disease,” is causing heavy losses in breeding herds of the UnitedStates and Canada. A similar disease has also appeared in much of Europeand the virus has been detected worldwide. The disease is manifested intwo forms, one causing severe reproductive failure in pregnant sows,manifested in the form of premature farrowings, increased numbers ofstillborn, mummified and weak-born pigs, decreased farrowing rate, anddelayed return to estrus and the other producing respiratory distress inpigs evidenced by lesions that appear in the lungs of infected swine.

The reproductive form of the disease is described by Keffaber, K. K.,“Reproductive Failure of Unknown Etiology”, American Association ofSwine Practitioners Newsletter, 1:109 (1989). The most prominentclinical symptoms of the reproductive form of the disease arespontaneous late-term abortions, premature births (which can be as highas 20–30% of all births) and the farrowing of mummified fetuses,stillborn or sickly piglets. Such clinical symptoms will typically beobserved in a herd from 4–16 weeks, or even longer. Stillborn fetuses inaffected litters often are in the early stages of mummification, asevidenced by tan-brown discoloration of the skin and post-mortemautolysis. Dome-shaped malformations of fetal skulls is also sometimesseen. The infection of sows may go unnoticed, or may manifest itself byan impaired general condition lasting up to a few days. For example, thesows may go off feed, and experience body temperatures either above orbelow normal. In the farrowing phase, the sows may exhibit depression,lethargy, pyrexia and occasional vomiting. In some affected herds, up to75% of all piglets may be lost. The economic consequences of thedisease, accordingly, are devastating.

The respiratory form of the disease exhibits clinical signs which aremost pronounced in piglets of 3–8 weeks in age, but are reported tooccur in pigs of all ages in infected herds. The diseased piglets growslowly, have roughened hair coats, respiratory distress (“thumping”) andincreased mortality (up to about 80% pre-weaning mortality). To combatthe problems associated with infection by PRRSV, vaccines have beendeveloped in an attempt to confer immunity to the current PRRSV strains.The present vaccines are only marginally effective and are all producedin cell lines of simian origin which possess a risk of continuousintroduction of primate viruses in swine populations. Findings inpreliminary studies of gross and microscopic lesions of piglets affectedwith the respiratory form of the disease suggest that microscopic lunglesions are an important clinical feature of this disease.

PRRSV belongs within the order Nidovirales in the family Arteriviridae.Other members in the family include equine arteritis virus, lactatedehydrogenase elevating virus, and simian hemorrhagic fever virus.Currently, there is no known human arterivirus. The PRRSV genome is apositive sense RNA about 15.1 kb in length. Untranslated regions (UTR's)of 156–220 nucleotides at the 5′ end and 59–117 nucleotides at the 3′end flank the viral genome. The viral genome has eight overlapping openreading frames encoding functional and structural proteins. PRRSV growsprimarily in the macrophages of infected pigs. In cell culture, thevirus is known to grow in CL 2621, MA-104, MARC cell lines and inprimary cultures of porcine alveolar macrophages. All of thesecontinuous cell lines are of simian origin and pose a risk ofintroduction of primate viruses into swine populations. Entry of thevirus occurs by receptor-mediated endocytosis and the receptor has beencharacterized as a heparin-like molecule. However, the mechanisms of howviral RNAs enter after endocytosis into the cell cytoplasm are notknown.

Replication of arteriviruses is similar to that of the coronaviruses.Genomic and subgenomic (−) sense mRNAs are formed in the infected cellsalong with the (+) sense mRNAs. Subgenomic (−) sense mRNAs have beenshown to function as the principal templates for mRNA synthesis incoronaviruses. Discontinuous transcription occurs in arteriviruses withthe formation of a functionally monocistronic, 3′-coterminal, nested setof mRNAs. The common leader is joined to the coding region by consensusintergenic sequences through the junction sequence UCAACC. In mousehepatitis virus (MHV), a corona virus, interaction of the leader,intergenic sequence and the body of the RNA involves cis and transacting elements. The 3′UTR's of MHV, coxsackie-, rhino- and polioviruseswere shown to be essential for the transcription of the genome. There islittle sequence complementarity between the 3′UTR and upstreamregulatory sequences for interaction, therefore it may be mediatedthrough RNA-protein-RNA interactions involving the viral or cellularproteins.

There are numerous studies on the interactions of viral and cellularproteins with 5′ and 3′ UTR's of viruses. Sindbis virus, brome mosaicvirus, QB phage and polioviruses require the interaction of certain hostcell proteins with viral UTR's for transcription to proceed. Althoughnumerous proteins have been shown to bind to these regulatory regions,only a few of them have been characterized. La protein, poly (rC)binding protein 2, and polypyrimidine tract binding protein are shown tobind to UTR's of poliovirus. Polypyrimidine tract binding protein andheterogeneous nuclear ribonucleoprotein have been shown to bind to theleader sequence of MHV. These proteins are predicted to play a role inmRNA splicing and transportation. Some cytoskeletal and chaperoneproteins, like actin, tubulin, and heat shock proteins, are shown tohave RNA binding activity. These proteins might play a structural rolein viral RNA synthesis or in orienting and transporting the RNAreplication complexes to the site of replication.

The study of host cell proteins that interact with viral RNAs is stillin the infancy stage and there is a lack of important informationregarding this interaction, especially with respect tohost-susceptibility factors. Even in arteriviruses, such as PRRSV, thehost susceptibility factors have not been studied. Thus, the markers forswine breeding for increased host susceptibility to PRRSV are not known.However, it is known that different breeds of pigs do differ in PRRSVsusceptibility based on experimental infection followed by sacrificingthe animals followed by further examination with histopathology andimmunohistochemistry for interstitial pneumonia and presence of PRRSVantigen in the lungs.

Moreover, only simian cell lines provide the cell culture for currentPRRSV vaccines which is a dangerous activity. The use of simian celllines for these cell cultures might accidentally introduce primateviruses of significance into swine lines intended for xenotransplantpurposes. Because swine are being increasingly explored as a source ofxenotransplanted organs to meet the shortage of organ transplants forhumans, the introduction of primate cell lines to swine populations mayultimately pose a risk to humans having xenotransplanted organs. Thus itwould be prudent to avoid the use of simian cell lines in swine vaccinepreparations.

Accordingly, what is needed in the art is a method of screening swinefor susceptibility to PRRSV infection. Preferably, this screening testis DNA-based and is able to correlate the sequence variation in criticalmotifs or domains of the susceptibilty gene with low, medium, or highdegrees of susceptibility to PRRS. Still more preferably, this screeningshould be non-invasive and able to be performed on a number of differentanimal fluids and cellular material, including swine germplasm and wholeblood. Still more preferably, this screening is performed on ear notchbiopsies for detecting pigs with lower susceptibility to PRRS.Additionally what is needed is a method of using these screening resultsin a breeding program designed to lessen the susceptibility of offspringto PRRSV infection. Another need in the art is to identify singlenucleotide polymorphisms (SNPs) in DNA which impact and correlate withsusceptibility resistance to PRRV infection. What is further needed isan understanding of the interaction between genes impacting on PRRSVsusceptibility and regulatory elements and/or other genes (cis- andtrans-acting factors) affecting the expression and regulation of thegenes which are related to PRRSV susceptibilty. What is still furtherneeded is an understanding of the chromosomal organization and locationof the gene. What is further needed is a non-simian cell line forpropagating high-titer PRRSV vaccine stock to avoid crossover of primateviruses into the swine population. Such a cell line will be especiallyadapted for vaccines used with xenotransplanted swine. Finally, what isneeded is a class of compounds which can block the entry of viral RNAinto cells.

SUMMARY OF THE INVENTION

The present invention solves the prior art problems mentioned above andprovides a distinct advance in the state of the art. In particular,through the present invention, methods are provided which allownon-invasive genetic testing for PRRSV susceptibility, selection ofswine for further breeding, the development of high-titer vaccines innon-simian cell lines, the development of compounds which can be usedfor PRRSV treatment, and the detection and characterization of theinteraction between the 3′UTR RNA of the PRRS genome and susceptiblecell lines, thereby leading to the development of novel anti-viralcompounds called anti-RNA Entry Compounds. Additionally, the presentinvention defines the genomic sequence and organization of porcine CD151, demonstrates the RNA-binding activity of porcine CD 151, identifiesthe isoforms of porcine CD 151 in different tissues, and produces anon-simian cell line with a transfected porcine CD 151 gene. Finally,the present invention demonstrates the activity of the whole porcinemolecule and identifies the CD 151 domains that impart RNA-bindingactivity and confer susceptibility to the non-simian cell line, babyhamster kidney-12 (BHK-21).

Accordingly, one aspect of the present invention provides a method ofscreening swine using relatively non-invasive methods. For example, ablood sample could be drawn and the presence of CD 151 in plateletscould be detected using quantitative reverse transcriptase-polymerasechain reaction (RT-PCR) and/or DNA PCRs. Also, DNA PCRs can be performedon each notch biopsy or tail snip from piglets. The knowledge gainedfrom this type of screen could greatly improve swine breeding plans asswine with low levels or even no detectible CD 151 could be selected forfurther breeding. One example of a swine breeding program which utilizesadvantages of the present invention compares the CD 151 levels of anindividual swine to a known standard of CD 151. This known standard isgenerally found by comparing the CD 151 levels from a large number ofsamples of cellular material (e.g. fluid or tissue) from swine andrepresents the average level of CD 151 detected in the samples. Asdifferent cellular materials from the same animal may have differentlevels of CD 151, it is preferable to compare CD 151 levels from acellular material of known origin (blood, blood platelets, sperm cells,germplasm, semen, ova, etc.) with the known standard for that specificcellular material. Moreover, as the known standard represents an averageCD 151 level for swine, an individual swine may be selected for furtherbreeding based on their detected CD 151 level in comparison with theaverage. Generally, it is preferable to select swine with CD 151 levelswhich are lower than average as this would tend to produce offspringwith increased resistance to PRRSV infection. Other methods of screeningswine will include identifying desirable single nucleotide polymorphismsthat correlate with reduced susceptibility to PRRSV. In this manner,swine having certain levels of CD 151 which place the swine into aparticular desired percentile for CD 151 levels and which retain otherdesirable breeding characteristics would be selected for this furtherbreeding. Preferably, these swine would rank in the lower 50thpercentile for CD 151 levels. More preferably, these swine would rank inthe lower 80th percentile for CD 151 levels, still more preferably,these swine would rank in the lower 90th percentile for CD 151 levels,and most preferably, these swine would rank in the lower 95th percentilefor CD 151 levels. In other words, for this most preferred grouping, 95%of all swine would have higher CD 151 levels than the individual swinebeing tested. Of course, CD 151 deficient swine could be further bred toproduce a “knockout” line of swine which would be resistant to PRRSVinfection.

In a related aspect of the present invention, cellular material in theform of germplasm such as sperm cells, or extended porcine semen couldbe screened for the presence of CD 151 prior to distribution to breedingfacilities. Again, using the same general procedures described above,the knowledge gained from such a screening would be invaluable to futurebreeding plans and use of large amounts of germplasm. The ejaculationvolume in swine is approximately 500 mL and the semen gets furtherextended so even if the animal is no longer on the farm or has died, orhas been sold, the germplasm from a valuable boar can be used for manyyears and for many sows and could provide several future generations ofpiglets of desirable genotype or genetic makeup. Such frozen germplasmcan also be screened for CD 151 levels and PRRSV susceptibility beforeartificial insemination. This screening could also indicate thesusceptibility of animals or their offspring to infection by PRRSV ashigher levels of CD 151 are correlated with higher virus production andrelease by cells. Thus, these tests will also be applicable forscreening live animals on farms for their susceptibility to PRRSV. Theselection of swine for further breeding based on CD 151 levels detectedin germplasm would proceed as follows. A sample of germplasm would betaken and the amount or level of CD 151 in that sample would bedetermined by RT-PCR. This result (the amount of CD 151) would becompared with known standards for that sample as different types ofgermplasm would typically contain different amounts of CD 151. The knownstandards would be determined by the average of the quantitation of alarge number of such samples. Swine exhibiting reduced levels of CD 151in comparison to the standards (and thereby having a reducedsusceptibility to PRRSV infection) yet still possessing other valuablebreeding traits would be selected for further breeding. Preferably,these swine would have CD 151 levels lower than about 50% of all swinetested. More preferably, these levels will be lower than at least about80%, still more preferably, at least about 90%, and still morepreferably, at least about 95% of all swine tested. In other words, theCD 151 levels in the samples from these swine would be rankedapproximately in the bottom 20%, 10%, or 5% of all swine tested. Ofcourse, many variations of this breeding plan could result using themethods of the present invention and these variations are presumedcovered provided that the level of CD 151 in a sample of cellularmaterial is determined and this level of CD 151 influences the breedingstrategy.

The present invention also provides a method of ascertaining thesusceptibility to PRRS infection in animals. Such a method would includethe steps of obtaining a sample of cellular material such as tissue orfluid, performing a CD 151 assay on this sample, and using the resultsof this assay as a measure of the animal's susceptibility to PRRSinfection. The cellular material chosen for the assay can be anycellular material including any fluid or tissue or semi-purified orpurified cellular preparation which contains detectible levels of CD151. For example, blood, blood platelets, germplasm, sperm cells, ova,and semen all contain detectible levels of CD 151. One preferred assaywould include the steps of extracting the RNA from the sample and thenperforming RT-PCR on the extracted RNA. Preferably, the results wouldprovide a quantified amount of detected CD 151. This quantified amount(or level) could then be compared to a known standard for CD 151 levels,thereby indicating the susceptibility to PRRS infection for the animal.Still more preferably, the cellular material sample would be derivedfrom a known origin and the known standard would represent the knownstandard for cellular material of the same origin.

The present invention further provides a method for determining if ananimal is resistant to PRRSV infection based on the presence or absenceof CD 151 in cellular material. If CD 151 is absent, the animals willtypically be immune to PRRSV infection. If CD 151 is present the animalswill typically be susceptible to PRRSV infection to varying degreesbased on the actual CD 151 levels. In this manner, PRRSV infectionresistance in animals can be classified based on the detected level ofCD 151 in any sample of cellular material. Such a method would includethe steps of obtaining a sample of cellular material from the testedanimal, performing an assay on the sample to find the CD 151 level ofthat sample, comparing the CD 151 level of the tested animal with aknown scale of CD 151 levels wherein the known scale corresponds to aspecific degree of PRRSV infection resistance, and finally classifyingthe tested animal's PRRSV infection based on the comparison with theknown scale. Preferably, the sample of cellular material will be of aknown origin and the CD 151 scale will represent the scale for cellularmaterial from the same origin in other animals. Such a classificationmay result in a percentile ranking of PRRSV infection resistance in theanimal.

In another aspect of the present invention, in vitro diagnostic testsfor detection of the wild type virus as well as antibodies to the virusare developed. Such tests generally include the steps of obtaining asample of cellular material from an animal, performing a diagnostic testdesigned to detect either the antibodies to the virus or the virusitself in a recombinant cell line, and using the results of the testingto confirm the diagnosis of PRRS in an individual swine or in a swineherd. Preferred diagnostic tests include virus isolation assays andimmunodiagnostic assays such as ELISA, indirect fluorescent antibodytests, and indirect immunoperoxidase tests. Preferably, the recombinantcell line used will permit greater replication of the virus making thetest more sensitive to PRRSV infection or antibody presence.

In another aspect of the present invention, higher titer PRRSV vaccinescould be developed in CD 151 transformed cell lines to aid in theimmunization of swine herds. In a related aspect of the presentinvention, other non-simian cell lines can be transformed with CD 151and used to propagate high titer PRRSV vaccine stocks. This has becomemore of an issue as xenotransplantation (especially in swine) becomesmore developed. Use of vaccines made in simian cells may transmit thesimian viruses to swine, and hence, organs used for xenotransplantationmay also be susceptible to some of these primate viruses. Thus, it willbe much safer to avoid using the primate or monkey-kidney cell lines forPRRS vaccines and thereby eliminate the risk of subsequent primate virusintroduction into the human transplant recipient population. For celllines which are already susceptible to PRRS infection, transformationresulting in even higher production of CD 151 can be accomplished usingmethods of the present invention.

In another aspect of the invention, non-simian cell lines aretransfected with non-simian CD 151 in order to produce a transformedcell line. In this respect, it is preferred to transform non-simiancells with non-simian CD 151 in order to completely avoid primate virusintroduction into the human transplant recipient population. Aparticularly preferred non-simian CD 151 sequence is provided as SEQ IDNo. 14 which represents the first complete genomic sequence of porcineCD 151. Preferably, sequences having at least 84% sequence homology withthis sequence will be used to transform cells for purposes of thepresent invention. More preferably, the homology to SEQ ID No. 14 willbe at least 90% and still more preferably, at least 95%. Mostpreferably, the homology to SEQ ID No. 14 will be at least 98%.

Another related aspect of the present invention will be that the hightiter vaccines propagated in non-simian cell lines can be used for otherapplications such as development of diagnostic tests for detection ofantibodies against PRRS, such as an ELISA assay.

Another related aspect of the present invention is the development anduse of a plasmid or vector capable of transforming cell lines andenhancing their susceptibility to PRRSV infection. A particularlypreferred plasmid in this respect has been given the designation pKSU(Genbank Accession Number AF 275666), and contains the sequence which isprovided herein as SEQ ID No. 1 and is also described in FIG. 1.Preferably, sequences having at least about 91% sequence homology or 93%sequence identity with SEQ ID No. 1 are embraced by the presentinvention, whether the sequence appears as an isolated sequence, in aplasmid, or in another suitable vector. More preferably, such sequenceswill have at least about 95% sequence homology or 97% sequence identityand still more preferably at least about 98% sequence homology or 99%sequence identity with SEQ ID No. 1. It is believed that this sequencerepresents the first reported isolated simian CD 151 sequence. Ofcourse, the corresponding amino acid sequences for this and similarsequences are also presumed covered by the present invention as theirdetermination requires no more than routine skill in the art. When it isdesired to use non-simian CD 151 in the development of a vector orplasmid for use in transforming cell lines, sequences having at least84% sequence homology with SEQ ID No. 14 are preferably selected forinclusion in the plasmid or vector. More preferably, the sequenceselected will have at least 90% sequence homology and still morepreferably at least 95% homology. Most preferably, the homology will beat least 98% in comparison to SEQ ID No. 14.

Similarly, the present invention provides a method for incorporating CD151 coding sequences directly in the genome of an animal. This methodgenerally includes the step of integrating the sequence of interest(e.g. CD 151) into the chromosome using a vector designed for such aninsertion. In other words, the sequence of interest is incorporated intoa retro-viral vector and this retro-viral vector is used to integratethe sequence directly into a chromosome in the genome.

Accordingly, methods for preparing PRRSV vaccine stock are provided bythe present invention. In general, to prepare PRRSV vaccine stock, acell line is provided and then transformed with CD 151. This cell linecan be resistant to PRRSV infection, or can be susceptible to PRRSVinfection prior to transformation. The resultant transformed cell lineis then infected with PRRSV and caused to produce PRRSV progeny for usein the vaccine stock. As noted above, the cell line is preferably ofnon-simian origin. In this manner, cell lines which are previously notsusceptible to PRRSV infection are rendered susceptible aftertransformation with CD 151 and cell lines which were previouslysusceptible to PRRSV infection produce much greater numbers of progenyvirus. Preferably, the transforming step includes stable transfectionwith a plasmid containing a CD 151 DNA sequence. The CD 151 sequence canbe derived from any animal which has CD 151. Preferably the CD 151 isporcine or simian CD 151. When the CD 151 DNA is of or desired to be ofporcine origin, the sequence should have at least 84% sequence homologywith SEQ ID No. 14. More preferably, this sequence homology should be atleast 90% and more preferably 95%. Most preferably, when the CD 151 DNAis or desired to be of porcine origin, the CD 151 should have at least98% sequence homology with SEQ ID No. 14. When the CD 151 DNA is ordesired to be of simian origin, the CD 151 DNA sequence will have atleast about 91% sequence homology with SEQ ID No. 1. Still morepreferably, the CD 151 DNA sequence has at least about 95% and stillmore preferably at least about 98% sequence homology with SEQ ID No. 1.The resultant vaccine stock is of higher titer than was previouslyobtainable prior to transformation. In some cases, this vaccine stock'stiter is about 100 fold higher than was previously possible.

In another aspect of the present invention, polymorphisms in CD 151sequences are detected and identified using RT-PCR followed bysequencing or a specifically designed test. These differentpolymorphisms are then analyzed to determine their effects on PRRSVsusceptibility. The use of SNPs in this regard is very helpful and suchmethods have gained wide acceptance for determining diseasesusceptibility and the evaluation and selection of breeding swine.Moreover, the level of CD 151 may be affected by regulatory sequences,such as promoters of the gene. The regulatory sequences are thenanalyzed to know tissue specific differences and differences amongindividual piglets. Additionally, the information obtained from thechromosomal localization and localization of the gene can be used toselect the lines of pigs for disease resistance. Of course, collateraleffects on important production traits should be monitored in order toreach a balance between any positive and negative effects that occurfrom the selection for disease resistance traits.

Still another aspect of the present invention is the development and useof anti-RNA Entyr Proteins (anti-REPs) by the Northwestern strategy.Anti-REPs will be an important addition to the arsenal of drugs againstemerging viruses. It is now believed that novel emerging viruses willall be RNA and most likely will emerge in wildlife populations due toencroachment of humans to wildlife habitats. These anti-REPs areextremely useful because they provide a readily available strategy thatcan be used for viral diseases that may emerge in animals (bothdomesticated and wild) and humans in future. It is important tounderstand the identification and mechanisms of RNA-binding proteinssuch as CD 151 for PRRSV and discovery of the compounds belonging to thecategory of anti-REPs. One potential method for blocking the entry ofPRRSV into cells includes the step of blocking PRRSV viral RNA frominteraction with CD 151. This blocking is preferably accomplished bycontacting the cells with an anti-viral compound, preferably an anti-REPcompound. These anti-viral compounds will be designed to occupy bindingsites on CD 151, thereby blocking this viral RNA-CD 151 interaction.Preferably, these anti-viral compounds will have a greater affinity aswell as a greater avidity for these binding sites than the PRRSVviral-RNA and should be producible in a high throughput manner.

Another aspect of the present invention is the ability of CD 151 to bindto RNA and this is the first report of a tetraspan molecule havingRNA-binding activity. This discovery can be used to develop a novelclass of compounds that prevents PRRSV or viral RNA-binding to thistetraspan and thus being released into the cell cytosol from theendosome. Combinatorial chemistry and screening by a high throughputscreening system can be used to find lead compounds to treat PRRSV andpotent anti-REP compounds can be found by a modified Northwestern assay.

With respect to porcine CD 151, the present application defines thegenomic sequence of porcine CD 151 and determined that porcine CD 151mRNA has about 84% sequence homology with known CD 151 sequences frommonkeys, mice, rats, and humans. However, it was determined that theintron portions of porcine CD 151 shared little to no homology withother known CD 151 intron sequences. The sequence of porcine CD 151 isprovided herein as SEQ ID No. 14. Due to the lower homology sharedbetween porcine CD 151 and the other known sequences of CD 151,sequences having greater than 84% sequence homology are consideredcovered by the present invention as no known CD 151 has such a highdegree of homology. Preferably, the sequence used to practice thepresent invention will have at least 90% or even more preferably atleast 95% sequence homology with SEQ ID No. 14. Most preferably,selected sequences will have at least 98% sequence homology with SEQ IDNo. 14. Furthermore, when constructing CD 151, it is preferred to useexons and introns which have similar homologies to sequences listedherein as SEQ ID Nos. 15–29. In other words, it is preferred that allportions of the CD 151 sequence share a certain degree of homology withSEQ ID Nos. 15–29. In the case of the exons of CD 151, the homology ispreferred to be at least 84% for each of the discrete exons listed asSEQ ID Nos. 15, 17, 19, 20, 22, 24, 26, and 28. More preferably, thehomology is preferred to be at least 90% and still more preferably, atleast 95%. Most preferably, each CD 151 sequence should containsequences which have at least 98% sequence homology with each of SEQ IDNos.15, 17, 19, 20, 22, 24, 26, and 28. In the case of introns, thehomology need not be so high as the porcine CD 151 introns shared nohomology with any known sequences.

It was also determined that porcine CD 151 is expressed at high levelsin heart, muscle and endothelium. Furthermore, methods are providedwhich permit for the screening for porcine CD 151 in pigs by a moreuser-friendly PCR test on ear notch biopsies or semen and ova samples.

Porcine CD 151 was also shown in a northwestern assay to have RNAbinding activity with the 3′-UTR of PRRSV and that this activity islocalized in the exodomains of CD 151 exodomain 1 and exodomain 2 byNorth-Western mobility shift assays. Finally, the present inventiondemonstrates that the amount and isoform of porcine CD 151 variesbetween different porcine tissues depending upon the type of tissue, theage of the pig, and the breed of the pig.

PRRSV is a positive sense RNA virus causing serious economic losses inswine. Previous studies have shown that 3′UTR RNA of the arterivirusesplays an important role in the replication of the virus through theinteraction with the cellular proteins. A cDNA library of MARC cells wasconstructed in the λ ZAP Express vector and the library was screenedwith positive sense 3′UTR ([α-³²P] UTP) RNA of PRRSV. A RNA bindingclone with an insert of 1.4 kb was found, and, after sequencing, itexhibited homology to CD 151, a transmembrane protein belonging to thetetraspan family of proteins. The MARC and BHK-21 cells were transfectedwith the CD 151 plasmid, and the fusion protein was immunoprecipitatedwith anti-Lac Z and anti-CD 151 antibodies. On Northwestern blotting,the precipitated 29 kD protein interacted with the radiolabelled 3′UTR.The precise function of CD 151 is not known, but it has been shown toplay roles in cell-cell adhesion, regulation of vascular permeability,and transmembrane signaling. The BHK-21 cell line lacks CD 151 and isnot susceptible to PRRSV infection, but when the BHK-21 cell line wastransfected with the CD 151 plasmid, it became susceptible to PRRSVinfection. Similarly, the non-simian cell lines can be transformed withCD 151 and used for PRRSV propigation to high titer.

In order to identify cellular proteins that bind to 3′UTR of PRRS virus,RNA ligand screening of a MARC cell expression library was performed.Moreover, this is the first report of the RNA binding property of atetraspan molecule, Platelet Endothelial Tetraspan Antigen-3 (PETA-3),also designated as CD 151, and its role in PRRS virus infection. It isknown that viruses bind to receptors (protein-protein interaction) andget into the cell in endosome. Under low pH conditions, the virusundergoes structural disorganization and releases viral RNA into theendosome. However, the method of entry of viral RNA in endosome intocell cytoplasm is not known. The present invention demonstrates that RNAbinding proteins, such as CD 151, help PRRSV to enter into cellcytoplasm for further replication of the virus (FIG. 9).

The present invention also determined the mRNA sequence for porcine CD151 and this sequence is provided herein as SEQ ID No. 38. For purposesof the present invention, it is preferred to use mRNA sequences whichcorresponds to a DNA sequence having at least 84% sequence homology withSEQ ID No. 38. More preferably, the DNA sequence will have at least 90%and still more preferably at least 95% and most preferably at least 98%sequence homology with SEQ ID No. 38.

The present invention also provides the DNA sequence coding for CD 151.When using DNA sequences coding for CD 151 which are non-simian inorigin, it is preferred that such sequences are derived from porcine CD151 and have at least 84% sequence homology with SEQ ID No. 14. Morepreferably, the DNA sequence will have at least 90%, more preferably atleast 95%, and most preferably at least 98% sequence homology with SEQID No. 14. The genomic sequence of porcine CD 151 is the combination ofintrons and exons summarized in Table 2. As noted above, the exons share84% or less sequence homology with other known CD 151 sequences and theintrons share no homology with any known sequences. Accordingly, when itis desired to construct or use a CD 151 sequence of non-simian origin,the DNA sequence is preferred to have at least one of the introns haveat least 84% sequence homology with a sequence selected from the groupconsisting of SEQ ID Nos. 16, 18, 21, 23, 25, 27 and 29. Similarly, theDNA sequence is preferred to have at least one of the exons with atleast 84% sequence homology with a sequence selected from the groupconsisting of SEQ ID Nos. 15, 17, 19, 20, 22, 24, 26, and 28.

In another aspect of the present invention, a vaccine for inducingeffective immunity against PRRSV is provided. The vaccine comprisesviral progeny produced in a non-simian cell line transformed withnon-simian CD 151. Preferably, the CD 151 is of porcine origin. Stillmore preferably, the CD 151 will have at least 84% sequence homologywith SEQ ID No. 14.

In another aspect of the present invention, a method of determining theeffect of single nucleotide polymorphisms on PRRSV susceptibility isprovided. The method generally comprises the steps of obtaining at leasttwo CD 151 genomic sequences, comparing single nucleotide polymorphismsbetween the sequences, and correlating the single nucleotidepolymorphisms with susceptibility to PRRSV.

Finally, the present invention provides a method of comparing PRRSVsusceptibility factors between individual swine, the method generallycomprises the steps of obtaining the genetic sequence of at least twoswine, analyzing the amount of CD 151 encoding sequences, and comparingthe amount of CD 151 encoding sequences. The genetic sequence of theswine can be obtained from a biopsy from an ear notch or tail snip, andperforming PCR on the biopsy to determine CD 151 sequences. Informationobtained using such method can also be used to correlate expressionlevels with susceptibility to PRRSV.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is the nucleotide sequence of the simian CD 151 cDNA clone, andthe predicted amino acid sequence wherein the putative transmembranedomains are underlined and the inframe stop codon is indicated by anasterisk;

FIG. 2 is a photograph of the RNA-binding activity of the CD 151 proteindetected by immunoprecipitation with CD 151 mAb in a Northwesternblotting assay;

FIG. 3 is a photograph of the RNA binding activity of the CD 151 proteindetected by immunoprecipitation with β-galactocidase mAb in aNorthwestern blotting assay;

FIG. 4 is a photograph of the in vivo RNA binding activity of the CD 151protein, as demonstrated by RT-PCR after immunoprecipitation of PRRSVinfected MARC cell lysates;

FIG. 5 is a photograph of the RT-PCR result illustrating theamplification of the CD 151 105 bp amplicon;

FIG. 6 is a photograph of the results of a western blotting experimentdetecting the presence of CD 151;

FIG. 7 a is a photograph of immunohistochemistry staining to detect thepresence of PRRSV in wild-type BHK-21 cells showing no detection ofPRRSV antigen;

FIG. 7 b is a photograph of immunohistochemistry staining to detect thepresence of PRRSV in BHK-21 cells which have been transfected with CD151;

FIG. 8 is a graph illustrating the enhanced PRRSV production by MARCcells after stable transfection with CD 151;

FIG. 9 is a schematic representation of the mechanism of PRRS viral RNAentry into a target cell and the role of RNA-binding proteins such as CD151;

FIG. 10 is a schematic representation of the sequence of porcine CD 151DNA; and

FIG. 11 is a photograph of a northwestern blot illustrating the RNAbinding activity of porcine CD 151.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following examples set forth preferred embodiments of the presentinvention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

As used herein, the following definitions will apply: “SequenceIdentity” as it is known in the art refers to a relationship between twoor more polypeptide sequences or two or more polynucleotide sequences,namely a reference sequence and a given sequence to be compared with thereference sequence. Sequence identity is determined by comparing thegiven sequence to the reference sequence after the sequences have beenoptimally aligned to produce the highest degree of sequence similarity,as determined by the match between strings of such sequences. Upon suchalignment, sequence identity is ascertained on a position-by-positionbasis, e.g., the sequences are “identical” at a particular position ifat that position, the nucleotides or amino acid residues are identical.The total number of such position identities is then divided by thetotal number of nucleotides or residues in the reference sequence togive % sequence identity. Sequence identity can be readily calculated byknown methods, including but not limited to, those described inComputational Molecular Biology, Lesk, A. N., ed., Oxford UniversityPress, New York (1988), Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey (1994); Sequence Analysis in Molecular Biology, vonHeinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov,M. and Devereux, J., eds., M. Stockton Press, New York (1991); andCarillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), theteachings of which are incorporated herein by reference. Preferredmethods to determine the sequence identity are designed to give thelargest match between the sequences tested. Methods to determinesequence identity are codified in publicly available computer programswhich determine sequence identity between given sequences. Examples ofsuch programs include, but are not limited to, the GCG program package(Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)),BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol.,215:403–410(1990). The BLASTX program is publicly available from NCBIand other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403–410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 95% “sequence identity” to a reference nucleotide sequence, itis intended that the nucleotide sequence of the given polynucleotide isidentical to the reference sequence except that the given polynucleotidesequence may include up to 5 point mutations per each 100 nucleotides ofthe reference nucleotide sequence. In other words, in a polynucleotidehaving a nucleotide sequence having at least 95% identity relative tothe reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving a given amino acid sequence having at least, for example, 95%sequence identity to a reference amino acid sequence, it is intendedthat the given amino acid sequence of the polypeptide is identical tothe reference sequence except that the given polypeptide sequence mayinclude up to 5 amino acid alterations per each 100 amino acids of thereference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 95% sequence identity with areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total number of aminoacid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or the carboxy terminal positions of the referenceamino acid sequence or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence or in the one or more contiguous groups within the referencesequence. Preferably, residue positions which are not identical differby conservative amino acid substitutions. However, conservativesubstitutions are not included as a match when determining sequenceidentity.

Similarly, “sequence homology”, as used herein, also refers to a methodof determining the relatedness of two sequences. To determine sequencehomology, two or more sequences are optimally aligned as describedabove, and gaps are introduced if necessary. However, in contrast to“sequence identity”, conservative amino acid substitutions are countedas a match when determining sequence homology. In other words, to obtaina polypeptide or polynucleotide having 95% sequence homology with areference sequence, 95% of the amino acid residues or nucleotides in thereference sequence must match or comprise a conservative substitutionwith another amino acid or nucleotide, or a number of amino acids ornucleotides up to 5% of the total amino acid residues or nucleotides,not including conservative substitutions, in the reference sequence maybe inserted into the reference sequence.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size,hydrophobicity, etc., such that the overall functionality does notchange significantly.

Isolated” means altered “by the hand of man” from its natural state.,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Cellular material” refers to cells, tissues, and fluids containingmaterial of cellular origin.

“Anti RNA Entry Proteins” or Anti REPs” refer to a novel class ofchemicals or chemical analogues that prevent binding of the viral RNA,with an RNA protein. These Anti REPs are nontoxic to mammalian cells butare able to prevent infection of cells from RNA viruses.

EXAMPLE 1

This example identifies and describes the cells, virus and monoclonalantibodies used in later experiments, prepares the λ Zap expressionlibrary, details the cloning and probe preparation of the 3′UTR of thePRRSV genome, describes the screening of the cDNA library for cellscontaining CD 151, describes the transfection of cells with plasmidcontaining CD 151, and verifies that the transfection was successful andthat CD 151 binds to the 3′UTR of PRRS virus.

Materials and Methods:

The MARC, COS-7, Vero, CL 2621, MA-104 (derivatives of African greenmonkey kidney cells) and BHK-21 (Baby hamster kidney) cell lines wereused in the study. The MARC cells were grown in Eagle's minimumessential medium (MEM) (Life Technologies, Inc., Gaithersburg, Md.)supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, Utah).Dulbecco's minimum essential medium (Life Technologies, Inc.,Gaithersburg, Md.) with 10% FCS was used to cultivate BHK-21 cells. TheATCC VR 2332 strain of PRRS virus was used in the study. Unlessotherwise stated, the virus was propagated in MARC cells. The anti-CD151 monoclonal antibody (MAb) clone 14A2.H1 was purchased fromPharmingen International, San Diego, Calif. The anti-β-galactosidase MAbclone D19-2F3-2 (Boehringer Mannheim, Indianapolis, Ind.) and theanti-PRRSV nucleoprotein MAb, SR 30 (Rural Technologies, Brookings, S.Dak.), were used in the study.

To prepare the λ Zap expression library, the MARC cell line cDNA librarywas prepared using a ZAP Express cDNA synthesis kit (Stratagene, LaJolla, Calif.) following the manufacturer's instructions. Total cellularRNA was extracted from the cells by the acid phenol-guanidiniumisothiocyanate method. The mRNA was isolated from total cellular RNAusing an oligo(dT) cellulose column (Stratagene, La Jolla, Calif.), andthen 5 μg of mRNA was converted to cDNA and cloned directionally in theλ ZAP Express vector. The cDNA library was packaged using the ZAPExpress cDNA Gigapack III Gold cloning kit (Stratagene, La Jolla,Calif.). The quality of the library was verified by blue/white screeningand also by confirming the size of the insert.

To clone the 3′UTR of PRRSV genome and prepare a probe, the 3′UTR wascloned into the pCR II vector by RT-PCR amplification of the PRRSV RNAby TA cloning using the forward primer 5′-CCCCATTTTCCTCTAGCGACTG-3′ (SEQID No. 31) and the reverse primer 5′-CGGCCGCATGGTTCTCGCCAAT-3′ (SEQ IDNo. 32). The [α-³²P]UTP RNA transcript was prepared by in vitrotranscription. The cDNA was linearized by digestion with BamHI and thentranscribed in vitro using a Riboscribe™ (Epicentre Technologies,Madison, Wis.) T7 RNA synthesis kit and following the manufacturer'sinstructions. The DNA template was digested with 1 MBU of RNase-freeDNase for 15 minutes at 37° C. Quick Spin™ columns (Boehringer Mannheim,Indianapolis, Ind.) were used to remove free nucleotides. The probe wasprecipitated using 0.3 M sodium acetate and 2.5 volumes of 100% ethanolat −20° C. for 30 minutes. The activity of the probe was measured with aradioactive counter (Bioscane, Inc., Washington, D.C.).

Next, the cDNA library was screened by Northwestern blotting aspreviously described with slight modifications. In all the rounds ofscreening, plaques were induced for 1.5 hours during the first lift and2 hours for the second with 10 mM IPTG. Nitrocellulose membranes fromplaque lifts were denatured in 6 M guanidinium hydrochloride for 30minutes and gradually renatured with equal changes of single bindingbuffer (SB Buffer: 15 mM HEPES [pH 7.9]; 50 mM KCl, 0.01% [v/v] NonidetP-40; 0.1% [w/v] Ficoll 400-DL; 0.1% [w/v] PVP-40; 0.1 mM MnCl_(2;) 0.1mM ZnCl_(2;) 0.1 mM EDTA; 0.5 mM DTT) every 10 minutes. Duringprehybridization and hybridization 250 μl of ssDNA and 5 μl (10 mg/ml)of yeast tRNA were added to 5 ml of SB buffer to block non-specificbinding. Hybridization was performed overnight with the 3′ UTR PRRSV[³²P]RNA probe at 500,000 cpm/ml. RNA binding activity was detected byautoradiography.

The corresponding positive plaques were cored and eluted in 1 ml of SMbuffer with 40 μl chloroform. The inserts were excised using theGigapack III gold cloning kit. The insert, now in the pBK-CMV vector,was sequenced manually with T7 and T3 primers using a Sequitherm EXCELII DNA sequencing kit (Epicentre Technologies, Madison Wis.). Sequencingwas also performed at the Iowa State University Sequencing Facility inAmes, Iowa. A BLAST homology search was carried out to identify similarproteins.

To transfect the BHK-21 and MARC cell lines, pBK-CMV plasmid containingCD 151 gene using Lipofectamine reagent following manufacturer'sinstructions (Life Technologies, Inc., Gaithersburg, Md.) was used. Fortransient transfection, cells were harvested 24 hours aftertransfection. Stable transfection was done by G418 (Omega Scientific,Inc., Tarzana, Calif.) selection in growth medium at a concentration of1 mg/ml for BHK-21 cells and 0.7 mg/ml for MARC cells. After selection,cells were maintained in the presence of G418 at 0.5 mg/ml for BHK-21cells and 0.35 mg/ml for MARC cells. The expression of CD 151 wasmeasured by immunoprecipitation.

Next, to demonstrate that CD 151 binds to the 3′UTR of PRRS virus, aNorthwestern blot of the immunoprecipitated CD 151 protein wasperformed. Transfected cells in 24 well plates were lysed in 100 μl ofsingle detergent lysis buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, PMSF100 μg/ml and 1% [v/v] NP-40). To 50 μl of cell lysate, 1 μl of Mab(anti-CD 151 and anti-β-galactosidase antibodies) was added and rockedovernight at 4° C. Immunocomplexes were precipitated on ice for 2 h withaddition of 4 μl of formalin-fixed S. aureus cells and centrifuged at4,000×g for 10 minutes. All subsequent washes were done bycentrifugation at 12,000×g for 10 minutes. The pellet was washed once incold TSA (0.05 M Tris-HCl [pH 8.0]; 0.15 M NaCl; 0.025% NaN₃), 1% TritonX-100 and 1% SDS. The second wash was done in cold TSA alone followed bytwo washes in 10 mM Tris-HCl [pH 7.5] containing 1 mM EDTA. The pelletwas suspended in 20 μl of 2×SDS loading buffer and electrophoresized ona 10% SDS-PAGE gel. The proteins were blotted on nitrocellulose membraneand the Northwestern blot was carried out as described above.

Results:

In order to identify the MARC cell proteins binding to 3′UTR RNA ofPRRSV, a cDNA library of MARC cells was made and screened byNorthwestern blotting using radiolabelled 3′UTR. The MARC cell cDNAlibrary had a titer of 10⁸ plaque forming units (pfu) with an averageinsert size of 1–4 kb (Data not shown). Approximately 6×10⁶ plaques werescreened by Northwestern blotting. The single reacting clone wasobtained by repeated plaque purification and rescreening five times. Inthe last round of screening, a single isolated plaque was cored andexcised. On restriction digestion, the size of the insert was found tobe 1.5 kb (data not shown). After manual sequencing, a BLAST searchshowed that the insert had 98% homology with CD 151/PETA-3, a tetraspanmolecule. Sequencing of the complete insert was also performed at theIowa State University Sequencing Facility in Ames, Iowa. The insert wasa full-length cDNA with start and stop codons and also a poly (A) tail.This is the first report of the complete sequence of a simian CD 151gene. The sequence (Genbank accession number, AF275666) is shown in FIG.1 with the putative transmembrane region underlined. Although thesequence provided is simian, it is believed that CD 151 sequencesisolated from other animal lines (e.g. swine) could also be used due totheir predicted homology with this simian CD 151 sequence. The presenceof porcine CD 151 in alveolar macrophages has been proven by WesternBlot (see FIG. 5).

Immunoprecipitation followed by Northwestern blotting was carried out tovalidate the observation that CD 151 has RNA binding activity. BothBHK-21 and MARC cells were transfected with the pBK-CMV plasmidcontaining the CD 151 insert (obtained by library screening). Since CD151 was to be expressed as a lac Z fusion protein, the transfected celllysate was immunoprecipitated with both anti-CD 151 andanti-β-galactosidase MAbs. The protein immunoprecipitated with both MAbsexhibited RNA binding activity when probed with 3′ UTR PRRSV [³²P]RNA asshown in FIGS. 2 and 3 while the controls were negative. This provesthat the expressed protein, which is CD 151, has PRRSV RNA bindingactivity and that it binds to the 3′ UTR of PRRSV. In FIG. 2, lane 1contained the MARC lysate (without immunoprecipitation), lane 2contained transfected MARC cells, lane 3 contained transfected BHK-21cells, lane 4 contained untransfected MARC cells, and lane 5 containeduntransfected BHK-21 cells. For FIG. 3, lane 1 contained untransfectedBHK-21 cells, lane 2 contained transfected BHK-21 cells, lane 3contained transfected MARC cells, lane 4 contained untransfected MARCcells, and lane 5 of FIG. 3 is the MARC cell lysate(withoutimmunoprecipitation).

EXAMPLE 2

This example determined that CD 151 expression by BHK-21 cells renderedthe cells susceptible to PRRS infection.

Materials and Methods:

To determine whether BHK-21 cells expressing CD 151 (obtained by stabletransfection) become susceptible to PRRSV infection, immunohistochemicalstaining was performed. Cells were seeded in 24 well plates and infectedwith PRRSV at a multiplicity of infection (MOI) of 0.01 at 37° C. for 1hour. Infectivity was determined by immunohistochemistry 24-hours postinfection. Cells were fixed in acetone-PBS (3:1, v/v) for 10 minutes at4° C. and air-dried for 5 minutes. The primary antibody, anti-PRRSVnucleoprotein MAb (1:1000 dilution in PBS) was added and incubated for 1hour at 37° C. Cells were then washed once in PBS for 10 minutes andincubated with anti-mouse IgG biotinylated antibody (Vector Labs,Burlingame, Calif.) for 30 minutes at room temperature (RT). Afterwashing once in PBS for 10 minutes, avidin-biotin enzyme complex (VectorLabs) was added and incubated for 30 minutes at RT. The cells were thenwashed once in PBS for 10 minutes, once in distilled water for 5minutes, then DAB substrate was added and incubated in the dark for 10minutes. The cells were washed in distilled water for 5 minutes,counterstained with Gill's-1 hematoxylin for 30 seconds, washed in tapwater, then examined by light microscopy.

Results:

The results for this example are given in FIGS. 7 a and 7 b. The parentBHK-21 cell line pictured in FIG. 7 a was not susceptible to PRRSV butrecombinant CD 151-transformed BHK-21 cells pictured in FIG. 7 b werepositive for the PRRSV antigen, thereby indicating susceptibility toPRRSV infection. Thus, the introduction of CD 151 rendered a previouslyunsusceptible simian cell line susceptible to PRRSV infection. As shownin FIG. 2, untransfected MARC cells contain CD 151 while untransfectedBHK-21 do not contain CD 151. Interestingly, untransfected MARC cellsare susceptible to PRRSV infection while untransfected BHK-21 cells arenot.

EXAMPLE 3

This example utilized a virus burst assay to assess the effects of MARCcells which overexpress CD 151.

Materials and Methods:

The monolayer of MARC cells overexpressing CD 151 (obtained by stabletransfection as described in Example 1) was infected with PRRSV at a MOIof 0.1 at 37° C. for 1 hour. Cells were washed twice in MEM and overlaidwith 1 ml of MEM supplemented with 1% FCS. After incubating for 18hours, cells were lysed by freeze thawing and cell debris was removed bycentrifugation at 12,000×g for 5 minutes. The amount of virus in thesupernatant was titrated by plaque assay using parent MARC cells. Todetermine if there are any effects of overexpression of CD 151 on PRRSVreplication, MARC cells were examined with respect to the effect oninfectivity level. The MARC cells (both parent and transfected progenycell line overexpressing CD 151) were infected with equal amounts ofplaque purified PRRSV and allowed to grow for one complete replicationcycle (18 hours). The level of PRRSV was measured by plaque assay usingparent MARC cells. In plaque assay, 100 μl of serial dilutions (10-fold)of the supernatant were used for infection as described above. Afterinfection, the monolayer was washed once in PBS and once in MEM, thenoverlaid with 1 ml MEM containing 1% FCS and 1% agar. Plaques werevisualized after 1 day of incubation at 37° C. by staining with 0.01%neutral red.

Results:

There was an approximately 100-fold increase in the amount of virus intransfected MARC cells overexpressing CD 151 as compared to parent MARCcells and these results are given in FIG. 8. When these results aretaken in combination with the results from the previous experiment onviral entry, it is believed that CD 151 may have increased the levels ofPRRSV infection by promoting viral RNA or viral entry. Thus,introduction of CD 151 can be used to produce higher titer stocks ofPRRSV. Similarly, determining the amount or expression of CD 151 in aparticular swine will give an indication as to their susceptibility toPRRS infection, in comparison to a standard. In this manner, a largenumber of samples from different tissues and fluids will be assayed fortheir CD 151 levels in order to establish an average or standard forthat tissue or fluid. Individual levels of CD 151 obtained from the sametissue or fluid can then be compared to this average or standard inorder to determine an individual animal's susceptibility to PRRSinfection.

EXAMPLE 4

This example utilized RT-PCR to detect CD 151 mRNA in MARC cells.

Materials and Methods:

RNA was extracted from the PRRSV infected MARC cells using the acidphenol-guanidinium isothiocyanate method. RNA quality was evaluated byagarose gel electrophoresis. RT-PCR was performed using the GeneAmp RNAPCR kit (Perkin Elmer, Foster City, Calif.) with forward5′-CCTACCTGGCCACAGCCTAC-′3 (SEQ ID No 33) and reverse5′-ACAGGCGCAGCAGGTTCCGA-′3 (SEQ ID No 34) primers. The reversetranscription reaction was performed at 42° C. for 45 minutes, 95° C.for 10 minutes and 5° C. for 5 minutes. PCR was done at 94° C. for 2minutes, 94° C. for 30 sec, 55° C. for 30 sec, 72° C. for 15 sec, for 25cycles and 72° C. for 30 minutes. The products were analyzed in 2%agarose gels stained with ethidium bromide.

Results:

A predicted RT-PCR result showing the amplification of the 149 bpamplicon is shown in FIG. 4. In this figure, the in vivo bindingactivity of CD 151 protein was demonstrated by RT-PCR afterimmunoprecipitation with CD 151 MAb of infected MARC cell lysates. Inthis figure, lane M is a 123 bp ladder, lane 1 is the PCR -ve control,lane 2 is the uninfected but immunoprecipitated MARC cell line, lane 3is the infected immunoprecipitated with unrelated wasp (Cotesia folepis)MAb, lane 4 is the infected MARC cells immunoprecipitated with CD 151MAb with no UV-crosslinking CL 2621 cell line; lane 5 is the infectedMARC cells immunoprecipitated with CD 151 MAb after 15 minutes of UVcrosslinking, lane 6 is the infected MARC cells immunoprecipitated withCD 151 MAb after 30 minutes of UV crosslinking, and lane 7 is theinfected MARC cells immunoprecipitated with CD 151 MAb after 45 minutesof UV crosslinking.

EXAMPLE 5

This example determined if CD 151 possessed in vivo RNA binding activityby utilizing in vivo cross-linking.

Materials and Methods:

MARC cell monolayers in 24 well plates were infected with PRRSV at a MOIof 0.1 at 37° C. for 1 hour. The monolayer was washed 3 times in PBS,twice in MEM, and replaced with MEM supplemented with 1% FCS. Eighteenhours post infection, the cells were washed twice in PBS then covered infresh PBS and held on ice in a UV cross linker (Fisher Scientific,Pittsburgh, Pa.) at a distance of 10 cm from the 300 λ light source tobe irradiated for 15, 30, and 45 minutes. After irradiation, the PBS wasremoved and the cells were lysed by addition of 50 μl of singledetergent lysis buffer then freeze thawed. Immunoprecipitation wasperformed as described above using anti-CD 151 MAb except that IgGcoupled sepharose beads were used instead of formalin-fixed S. aureuscells. Proteineous material was digested with Proteinase-K (4 μg/ml) for15 minutes at 37° C. and RNA was extracted using the phenol-guanidiniummethod. RT-PCR was performed to determine the presence of PRRSV RNAusing forward 5′TGGGCTGGCATTCTTGAGGC′3 (SEQ ID No 35) and reverse5′TTCGGGCCGCATGGT-TCTCGC′3 (SEQ ID No 36) primers.

Results:

UV cross-linking followed by RT-PCR was performed to determine if CD 151possesses in vivo RNA binding activity. The rationale was that if therewas any interaction between the CD 151 protein and the 3′ UTR RNA ofPRRSV inside the cell, the complex should immunopreciptiate with theaddition of anti-CD 151 MAb. The MARC cells were infected with PRRSV andto strengthen the interaction between 3′ UTR RNA of PRRSV and CD 151, UVcross-linking was performed as described in the methods.Immunoprecipitated complex was treated with proteinase K to remove theproteinaceous material. The 3′ UTR RNA of PRRSV was detected in theimmunoprecipitate by RT-PCR as shown in FIG. 4. The interaction betweenthe CD 151 protein and the 3′ UTR of PRRSV was strong and 15 minutes ofUV-induced cross-linking was sufficient to immunoprecipitate RNA alongwith CD 151 while the control with non-specific MAb was negative. Thus,CD 151 does possess in vivo RNA binding activity.

EXAMPLE 6

This example utilized western blotting to detect the CD 151 in MARC,BHK-21, and Vero cell proteins.

Materials and Methods:

MARC, BHK-21 and VERO cell proteins were electrophoresed in 10% SDS-PAGEgel and blotted onto a nitrocellulose membrane. After blocking overnightin 5% non-fat dried milk in PBS, the membrane was incubated in anti-CD151 MAb (1:2,000 in PBS-T) at room temperature for 5 hours. Themembranes were washed in PBS-T for 15 minutes twice and incubated inanti-mouse HRPO conjugate (1:10,000) for 2 hours at room temperature.After washing, the proteins were detected by 3, 3′, 5,5′-tetra-methylbenzidine (TMB) membrane peroxidase substrate followingmanufacturer's instructions (Kirkegaard and Perry Laboratories,Gaithersburg, Md.).

Results:

The presence of CD 151 in susceptible cells was determined by westernblotting using anti-CD 151 MAb. As shown in FIGS. 5 and 6, thesusceptible MARC cells have CD 151 protein while the non-susceptibleBHK-21 cells lack the protein. Moreover, transfected BHK-21 cells (FIG.5, lane 13) do have CD 151 and are susceptible to PRRSV infection. Therehas been no report of susceptibility of COS-7 cells for PRRSV, while inVero cells, the PRRSV enters the cells but does not result in productiveviral infection. The Vero cells might lack other cellular factorsrequired for PRRSV infection. These results indicate that CD 151 is oneof the factors in determining the susceptibility to PRRSV infection withpossible involvement of additional intracellular factors.

EXAMPLE 7

This example determined the correlation between the presence of CD 151and susceptibility to PRRSV infection.

Materials and Methods:

To determine the possible relationship between the presence of CD 151and susceptibility of to PRRSV infection, RT-PCR using CD 151 specificprimers was performed to screen susceptible and non-susceptible celllines. These cell lines included MA 104, MARC, Vero, COS-7, the three ofwhich are all derived from African green monkey cells, ST, BHK-21, MDBKand HRT (Human rectal tumor cells) cell lines. Total RNA was extractedas described in Example 4 and the presence of CD 151 was detected byRT-PCR using the procedures and primers described in Example 4.

Results:

In MA 104, MARC 145, Vero, COS-7, and ST cell lines there was a 105 bpamplicon which was of the expected size. There were additional higherbands in CL2621, Vero, and COS-7 cell lines that could be due to thepresence of alternate spliced forms of CD 151. The CD 151 was absent inBHK-21, MDBK (See FIG. 5) and HRT cell lines. Interestingly MARC 145, MA104, and CL2621 are the cell lines susceptible to PRRSV infection whileBHK-21 cell lines are non-susceptible. In the past, is was believed thatthe only cells that were susceptible to PRRSV were the simian cell linesCL-2621 and MARC-145 swine alveolar macrophages. Thus, the presentinvention refutes the previous claim that only simian cell lines aresusceptible to PRRSV (see U.S. Pat. No. 5,846,805) because aftertransfection with CD 151, which is an accessory factor, non-simian celllines are susceptible to PRRS virus. The BHK-21, MA 104, COS-7, ST,MDBK, and HRT 18 cell lines are available from the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852. Therepresentative accession numbers are: BHK-21 is ATCC# CRL8544; MA104 isATCC# CRL2378; COS-7 is ATCC# CRL1651; ST is ATCC# CRL1746; MDBK isATCC# CCL22; and HRT18 is ATCC# CCL244. The MARC 145 cell line wasobtained from National Veterinary Sciences Laboratory (NVSL) in Ames,Iowa and CL2621 is a proprietary cell line obtained from NVSL, Ames,Iowa. The Vero cell line has been accorded ATCC # CCL81. Thus, thisexample provides further evidence that CD 151 plays an important role inPRRSV infection.

EXAMPLE 8

This example determined the lack of direct protein-protein interactionbetween CD 151 protein and PRRSV proteins, thereby proving that CD 151is an accessory protein but not a PRRSV viral receptor.

Materials and Methods:

After establishing that CD 151 is essential for PRRSV infection, thepotential for direct interaction between the PRRSV proteins and CD 151was determined by coimmunoprecipitation. The infected MARC cells wereimmunoprecipitated with CD 151 MAb and the presence of PRRSV proteins inthe immunoprecipitate was detected by PRRSV polyclonal antibody as theprimary antibody and anti-swine HRPO as the secondary antibody followedby detection with the ECL western blot detection system (AmershamPharmacea Biotech, Piscataway, N.J.). The coimmunoprecipitation was alsoperformed by first immunoprecipitating with PRRSV polyclonal antibodyand checking for the presence of CD 151 in the immunoprecipitate by CD151 monoclonal antibody as the primary antibody and anti-mouse HRPO asthe secondary antibody.

Results:

There was no direct interaction between the CD 151 and PRRSV viralprotein. This eliminates the possibility of CD 151 as a receptor.However, it does not rule out the possibility of an indirect interactionbetween CD 151 and PRRS viral protein. The indirect interaction willinvolve identification of yet another protein that could fit andcomplete the interaction between CD 151 and PRRS viral protein. Thus,viral RNA-protein interaction has been validated but there is no directprotein-protein interaction between CD 151 and PRRSV viral proteins.Accordingly, CD 151 is an accessory protein but not a receptor proteinbecause it enhances PRRSV titers, and thus enhances and mediates theentry of viral RNA.

EXAMPLE 9

This example reviews the methods used to discover chemical compoundsused to stop the interaction between viral RNA and CD 151 and providefor a novel class of anti-viral compounds called anti-RNA entryproteins.

Materials and Methods:

Based on this invention it appears that entry of viral RNA into the cellis mediated by the interaction of the viral RNA with the RNA-bindingprotein. This test can be carried out easily in vitro. In ahigh-throughput system, the motif of the CD 151 or the full-length CD151 can be spotted with an automatic instrument on nitrocellulose or inany solid-binding matrix. In a negative control, where no drug ispresent, the binding aptamer of RNA in the form of a ssDNA can befluorescently labeled and subsequently added in a well. If no drug ispresent, a maximum level (100%) of fluorescence or binding activity willbe observed. A wide variety of unknown chemicals can be added to theplate. The compounds that block the binding of the oligonucleotide withthe RNA-binding aptamer will be further modified to find some effectivenon-toxic compounds that could be used to block the entry of PRRSV RNAin the cells. Similar principles can be used for the discovery oftreatments for a wide variety of animal viruses. It is believed thatthis novel class of pharmaceuticals has not been described in theliterature.

Alternatively, with the discovery of yeast hybrid systems 3′ UTR RNA ofPRRSV and CD 151 protein can be adapted to the yeast tri-hybrid systemfor discovery of compounds that will block their interaction.

EXAMPLE 10

This example shows a non-invasive method for the utilization of thepresent invention in screening live pigs and stored germplasm for CD 151mRNA and levels of susceptibility of possible progeny to PRRSV.Additionally, this Example is similar to Example 4 and illustratesapplications of this invention in screening live pigs boar semen andextends the application of the present invention to subsequent littersin future generations in swine breeding programs for improved swineresistance to PRRS virus.

Materials and Methods:

On the basis of this invention, it is clear that the level of CD 151 isa critical factor in determination of the susceptibility of the targetcell to PRRSV. Different breeding lines of pigs will be screened byquantitative RT-PCR for the level of CD 151 in cellular material such asalveolar macrophages, semen, germ cells, ova, and platelets. The linesof pigs having lower levels of CD 151 in target organs are expected tobe less susceptible to PRRS and are thus less affected by the harmfuleffects of PRRS infection. Currently, pigs are selected on the basis ofsacrificing after infection and are mated. The animals found to carryrelatively less amounts of CD 151 will be selectively mated against PRRSsusceptibility. This will allow a non-invasive method of checking theanimals. With the help of CD 151 knockout experiments, it will bepossible to develop a pig line that is completely resistant to PRRSvirus infection.

Results:

This example demonstrates that the present invention overcomes thecurrent limitation of sacrificing different lines of pigs fordetermining their susceptibility after experimental infection withPRRSV. Using non-invasive diagnostic methods such as peripheral bloodplatelets, stored germplasm, or extended and non-extended porcinegermplasm, swine breeding operations should be able to select foranimals that have reduced susceptibility to PRRSV infection.

EXAMPLE 11

This example permits quantification of CD 151 levels in different swinebreeding lines.

Materials and Methods:

This Example is similar to Example 4 except that the plasmid pKSU isused for quantitative purposes. This plasmid contains full-length openreading frames of CD 151. A known amount of this plasmid (e.g. 0.05micrograms) is introduced into the PCR tubes and known dilutions of thepredetermined sample will be added to cover the range of the expected CD151 levels in different swine tissues, thereby providing knownprequantified standards. The amount of CD 151 in unknown swine sampleswill be quantified by comparison with the known standard curve.Additionally, to find the precise amount of CD 151 in swine tissues, aquantitative TaqMan for CD 151 can be performed. This will provideprecise quantification of CD 151 in germplasm and peripheral bloodplatelets in picogram to nanogram amounts per milliliter or per gram ofknown swine tissue, per milliliter of germplasm, or per one millionswine platelets or per one million swine spermatazoa.

Results:

By quantitative CD 151 RT-PCR, swine breeding lines lower in CD 151 willbe selected for breeding for reduced PRRSV susceptibility.

EXAMPLE 12

This example illustrates the use of non-simian, recombinant cell linesfor production of higher titer, safer swine vaccines to control orprevent PRRSV.

Materials and Methods:

Based on this invention, non-simian cell lines transformed with CD 151are not only susceptible to PRRSV but propagate the PRRS virus to muchhigher titers than parent cell lines. First, the cell lines will bechecked for contaminations such as Mycoplasma, porcine parvovirus, andBVD virus using methods that are well known in the art. Once the cellline has been found to be free of these contaminates, the cell line willbe plated in flasks or roller bottles and will be infected with plaquepurified PRRS virus. The American isolate, Lelystad virus, or any otherPRRS virus isolate can be used. The cell cultures will be infected forsixty hours after which they will be frozen at −80° C., andfreeze-thawed 3 times. Cellular material will be removed and clearsupernatant will be used as a vaccine after titration. It is recommendedto use 1 million PFUs/ml and the dose of the vaccine will be 1 mlintramuscularly in the presence of the parenteral adjuvants. Thisvaccine can even be diluted because of its high titer for makingspecific doses per vial. Alternatively, various combinations that areknown in this field of art can be applied. All manipulations andmodifications included in this invention are routine in the field ofpreparation of biologics for animal vaccines.

Results:

The preceding materials and methods produce a safe, high titer killed ormodified-live virus vaccine for PRRSV in non-simian cell lines.

EXAMPLE 13

This example shows the application of non-simian cell lines forpropagation of PRRSV for development of in vitro diagnostic assays.

Materials and Methods:

Advantageously, non-simian cell lines can be used for propagating PRRSVbecause of their higher susceptibility for propagating the wild typevirus from clinical samples such as nasal swabs and tissues such aslung, lymph nodes, or pools of tissues from cases suspected of PRRSV,swine abortions, or respiratory diseases. These cells are plated tillmonolayers are confluent in approximately seventy-two hours, samples areinoculated, and the cell line is checked for the presence of virus by afluorescent antibody test using SDOW 17 FITC conjugated antibody. Thepropagated virus, as produced in Example 12, is used to coat ELISAplates and the standard procedures for development of ELISA technologyfor detection of PRRSV antibodies have been described using theprocedures of Witte et. al., Development of a RecombinantNucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantificationof Antibodies against Porcine Reproductive and Respiratory SyndromeVirus, 7 Clinical and Diagnostic Laboratory Immunology, 700–702, (2000),the content and teachings of which are hereby incorporated by reference.Currently the ELISA test is considered as the gold-standard formonitoring the swine PRRSV antibodies.

Results:

The wild type PRRS virus can be detected in vitro by virus isolation.Additionally, the presence and quantity of PRRS antibodies can bedetected using these immunodiagnostic assays. Other assays which wouldprovide in vitro diagnostic tests for detecting wild type PRRS virus andantibodies and verifying PRRS diagnoses in swine herds include indirectfluorescent antibody tests and indirect immunoperoxidase tests. Suchtests are well known in the art and could be developed using no morethan ordinary skill.

EXAMPLE 14

This example shows the application of immunodiagnostic tools, (e.g.quantitative ELISA) for CD 151 which can be used to monitor swine fluidsamples including platelet lysates, sperm lysates and swine alveolarmacrophage lysates.

Materials and Methods:

Protein is detected quantitatively using the methods of Witte et. al.,2000. For example, the level of CD 151 in swine samples can be detectedusing these methods. Samples will be freeze-thawed. The level of proteinwill be monitored.

Results:

This example permits the mass screening of swine samples using aquantitative CD 151 ELISA. Additionally, the amount of CD 151 proteinper ml of swine sample or per gram of tissue will be detectible usingthese methods.

EXAMPLE 15

This example illustrates that CD 151 from sources other than simiansources can be used to render a previously non-susceptible cell linesusceptible to PRRSV infection.

Materials and Methods:

A sample of cellular material containing CD 151 is obtained from aswine. The RNA is extracted from this sample as previously described andthen the porcine CD 151 is isolated using RT-PCR. The isolated CD 151 isthen deposited in an appropriate mammalian expression vector which canbe used to render previously non-susceptible cell lines susceptible toPRRSV infection. Examples of such cell lines include BHK-21.Additionally, by depositing the vector into previously susceptible celllines, these cell lines can be used to overexpress the CD 151.

Results:

Previously non-susceptible cell lines are rendered susceptible to PRRSVinfection after depositing a vector containing porcine CD 151 into thecell line. Previously susceptible cell lines can overexpress theinserted CD 151. Thus, CD 151 from any source can be used to transformcell lines including non-simian cell lines so that they express CD 151.This proves that, the source of the CD 151 (simian, porcine, etc.) isnot relevant to practice the invention and that all aspects of theinvention using simian CD 151 can also be accomplished using porcine orother similar CD 151 sequences.

EXAMPLE 16

This example proves that tetraspan CD 151 or its related homologues inother species such as equine CD 151 can be used to propagate theArteriviruses from equine and other species.

Materials and Methods:

A heterologous CD 151 sequence is isolated and used to make recombinantcell lines as previously described. This cell line is then used toisolate and propagate the virus as previously described.

Results:

Similar to PRRSV, other Arteriviruses can be isolated and propagated tohigh titers in homologous and heterologous cell lines includingnon-simian cell lines. Thus, other Arteriviruses can be approached inthe same manner as PRRSV in identifying methods of producing high titervaccines in cell lines which were previously resistant to infection bythe Arteriviruses and increasing virus production in cell lines whichwere previously susceptible to the Arteriviruses.

EXAMPLE 17

This example determined the DNA sequence and genomic organization ofporcine CD 151.

Materials and Methods:

In order to determine the entire sequence of porcine CD 151, primerswere designed in the end of each exon to the beginning of the next exonto determine the sequence of each intron. The primer pairs are shownbelow in Table 1.

Inter- SEQ genic ID Region Primer Pair Sequence Direction No 15′-AGCCTCCTCAGGAATTCTGTC-3′ Forward 2 5′-GAAGCAGCAGTTGAAGGTGA-3′ Reverse3 2 5′-TTTCACCTTCAACTGCTGCTTC-3′ Forward 4 5′-GCTGATGTAGTCGCTCTTG-3′Reverse 5 3 5′-GCCACAGCCTACATCCTAGTG-3′ Forward 65′-AGTAGACGTAGGCCAGGACTC-3′ Reverse 7 4 5′-TCCTGCTGCTGCTCATCTTTC-3′Forward 8 5′-CTCAGAGTGTCCTCCAGGTTCG-3′ Reverse 9 55′-TCAAGGCGAACCTGAAGGAC-3′ Forward 10 5′-TGTAGATGTTGGAGGCGTGG-3′ Reverse11 6 & 7 5′-CTGTGGCAGCAACAACTCC-3′ Forward 12 5′-CTCCAGCTTCAGGCTCTTGT-3′Reverse 13

The genomic DNA was extracted from the porcine alveolar macrophages (PAMcells) using a genomic DNA extraction kit and following themanufacturer's instructions (Dneasy Tissue Kit catalog # 69504, Qiagen,Valencia, Calif.). The PCR was set up as follows: Genomic DNA 200 ηg,10×PCR buffer, 2 mM MgCl₂, DMSO 2.5 μl, 10 nmol of each dNTP, and 20pmol of each primer. After initial denaturation at 95° C. for 5 minutes,2.5 units Taq polymerase (Perkin Elmer, Foster City, Calif.) was addedto the PCR mixture followed by 35 cycles at 95° C. for 30 seconds,annealing temperature specific for each intergenic region for 15seconds, extension temperature of 72° C. for 1 minute, with the finalextension at 72° C. for 45 minutes. The annealing temperatures for eachintergenic region were as follows: intergenic region 1—57° C.;intergenic region 2—58° C.; intergenic region 3—59° C.; intergenicregion 4—55° C.; intergenic region 5—53° C.; and intergenic regions 6 &7—57° C. The PCR products were electrophoresed onto 1% agarose gel andthe PCR band was excised and cloned to pGEM-T easy vector (Promega,Madison, Wis.). The sequence was then sequenced manually using aSequiTherm EXCEL II sequencing kit (Epicentre Technologies, catalog #SEM79100, Madison, Wis.) and following the manufacturer's instructions.

Results:

The sequence of porcine CD 151 is provided as SEQ ID No. 14. The intronsequence is known to vary between species as determined by sequenceanalysis of the human, rat, and mouse homologues of CD 151 and this isnow also know to be true for porcine CD 151 which has shown little to nohomology between the introns of porcine CD 151 and the introns of otheranimals which have CD 151.

The genomic organization of porcine CD 151 is presented in Table 2. Theintrons and exons are also provided with specific SEQ ID Nos as follows:SEQ ID No 15 is Exon 1; SEQ ID No 16 is Intron 1; SEQ ID No 17 is Exon2; SEQ ID No 18 is Intron 2; SEQ ID No 19 is Exon 3; SEQ ID No 20 isExon 4; SEQ ID No 21 is Intron 4; SEQ ID No 22 is Exon 5; SEQ ID No 23is Intron 5; SEQ ID No 24 is Exon 6; SEQ ID No 25 is Intron 6; SEQ ID No26 is Exon 7; SEQ ID No 27 is Intron 7; SEQ ID No 28 is Exon 8; and SEQID No 29 is Intron 8. All of the introns except for intron 3 areidentified below and the sequence of intron 3 would be determined usingmethods similar to those used to obtain the remaining intron sequences.Of course, it is possible that there is no intron 3 sequence. However,if Intron 3 exists, it can obtained using porcine genomic DNA.

TABLE 2 Exon 1 CGCCGCCACTGCCGGCCTCGGACGCGTGGACGAGCCTCCT SEQ ID No 15CAGGAATTCTGTCCACCTGTCTCAGAGAGGAGCGGTCCCC AGCAGCCAG Intron 1gtgagtgctgggcggggccgggtgaaagtccaccttggggcggtactgtctgaaagcgtggc SEQ ID No16 cccaggt gctgagcggggcctgggccgcactgccccctgtccctcagggtggacaccccaggacagagcgagcggggcacctctggggtgtccctggggaggagcagagcccgcggcggtgtttctccctggggttggtgtggctgtcctcaccggtaatgaggaccagcacgggaaggccccccaggcctgccacccgcccgccctggggtgggggggaaggaaggcaggcctgcccttgaacggaagcctgaggggcctcccggaggcccaggactaggctcgagagggcagcacagcctgtctgtgagtggccttctccccctcccctccagcagccgctaggttcctgccagcctgtgacgtggggcccggggaatgcttggggtggggtggggaagccttgcatagctcaccccgaaggtgcctgctggggcttcctaccctggctgccggcagggtgagtcagcgggtgacggctgggggtcctccacgcccccctcacggcccggggctctgggcagagaagctggcctgggggcagtgggccctccctcccttactggtactggggatgggggctcctgagctgcgctcccaggaagtgtcccccttcccacggacactcccagagccttcaccagccaactgggactggtctggatgtctgtgtcccctccttctgtccccactggcatcccccaggccggccctcccctgttccctgccccag Exon 2CCCAGGATGGGCGAATTCGGCGAGAAGGGCGCGCCA SEQ ID No 17TGCGGCCACCGTTTGCCTCAAGTACCTGCTTTTCACC TTCAACTGCTGCTTCTGG Intron 2gtgaggagtgggcccccgcttcccgagcccggggctcagcccgtctctcacacccgg SEQ ID No 18gccagacgtggtgattggtgtgcactgcccgcag Exon 3CTGGCTGGCCTGGCCGTCATGGCAGTGGGCATCTGG SEQ ID No 19ACGCTGGCCCTCAAGAGCGACTACATCAGCCTCCTGGCCTCGGGCACCTACCTGGCCACAGCCTACATCCTAGTGGTGGCGGGCATTGTTGTCATGGTGACCGGTGTTCTGGGCTGCTGTGCCACCTTCAAGGAGCGGAGGAACC TGCTGCGGCTG Exon 4TACTTCATCCTGCTGCTGCTCATCTTTCTGCTGGAGA SEQ ID No 20TCATCGCCGGAGTCCTGGCCTACGTCTACTACCAGCA G Intron 4gtgcgcggctggggcgggccgcaggggcgcgtacacgcacagggtgtgcacgcgc SEQ ID No 21atgagcacggacacgcacgtgccctgggaaggaagagatgcacacaggcagggacacgtgcacgcgaccccacaggttccatgctgaccgtcgtgtcgtcaggccctggaggtgggaccgtgtcaccagggaggacccccacaggggtggccagtctgcctgcagcacccaaccacccacctgggggccccagtgtgccctccctgcccccgaccccccaccccatgccatctgggtgacgttcccgggcacctgacccaccccaggcagcgtctgtctccagacctgtttggcaccttctgcgcctctcccgtctgtgcccatccccgcgctgccctcctccccgcttcttagtctttgtggctgaagctgtcgccacctcgccacag Exon 5CTGAGTGCAGAGCTCAAGGCGAACCTGAAGGACACT SEQ ID No 22CTGAGCAAGCGCTACCGCCAGCCGGGCCACGAGGGT GTGACCAGCGCCACGGATAAGCTGCAGCAGGAGIntron 5 gtcggtgggtggcgccgggcggcgtctcccctgacgtctgtcggagccctggctgctg SEQID No 23 cgggggttgtggggaggggacggggctcagcccgggccatgttcccgtgcag Exon 6TTCCACTGCTGTGGCAGCAACAACTCCCAGGACTGG SEQ ID No 24CGGGACAGTGAGTGGATCCTCTCGGGTGAGGCAGGCGACCGTGTGGTTCCTGACAGCTGCTGCAAGACGGTGGTGGCGGGCTGCGGGCGGCGGGACCACGCCTCCAAC ATCTACAAAGTGGAG Intron 6gtaggccagtgggggctgcacccgggatgctggaggcgggtgtccccgcacccgag SEQ ID No 25gggcccggggctggggaagaggccccccctgccctcgacgcgccgctcacccccac tcccaccccagExon 7 GGCGGCTGCATCACCAGGCTGGAGACCTTCATCCGG SEQ ID No 26GAGCACCTGAGGATCATCGGGGCTGTGGGCATCGGC ATCGCCTGTGTGCAG Intron 7tgcgggcgcggggcggcgcgggcgcggggcacggggcggcatgggcacaggcgc SEQ ID No 27gtgctcatgccggctcttggctcag Exon 8 GTGTTCGGCATGATCTTCACGTGCTGCCTGTACAAGASEQ ID No 28 GCCTGAAGCTGGAGCACTACTGA Intron 8ctgcccacggacctgggcccacgcctgcaccgcagcttctcaggatgcgctgactact SEQ ID No 29gacggctaggggctgcggtcccaggacgcggctcctcccctcccacactgccagggaggtggtgtcctcatgccagggcccacgaccgtgccatcaccgcgactcctggggaccgccaaccccagagggagcttcaagtgcctttcgctgccaccaaagtcccacggcccagcccggtccctcctgcctctggggtggctggggcttgagctcaaaccgtaaaggccccatgcccgctgccctctcctttggggtattgttccctgataacctggctccgtcacggggctggtctgtgccgagttaggcggaagctggccagcccggggccagtgccaggcggcaggcagcaggtgaggaagccgggtgcctgcctgctccaggaagagcaagagccctcccggcctggcctctgcgcccacttacctgctgctcccaccaccatctaaaggcccaggtggacggccacatgctgacgcccctccggggcagggcagccctctgggcctccttcactgctgtatccccatgcctaggactgctcctgctttgtgataggtgctcaataaacgcctgtggaccagaaaaaaaaaaaaaaaa

The discovered porcine CD 151 sequence was randomly matched with partialporcine CD 151 clones from deposited EST sequences in Genbank (Accessionnumbers: clone 1—BE233265; clone 2—AW314209; clone 3—AW786379) andcompared with the published genomic sequence of human CD 151. Until thepresent invention, these previously published random CD 151 partialsequences were used only for expression studies and therefore, thefull-length porcine CD 151 sequence had heretofore never beendetermined. Furthermore, these random partial EST sequences were notasociated with any known protein, gene, or function. It is alsoimportant to note that the use of the deposited EST sequences would notinherently lead one to the entire sequence of CD 151. This is especiallytrue in light of the fact that the intron sequences of porcine CD 151had no matches. Thus, this is the first report of the complete porcinemRNA and genomic sequences.

FIG. 10 is a schematic illustration of the porcine CD 151 sequence. Thenumbers 1–8 at the top of this figure represent the position of the CD151 exons. Exons are also represented by the vertical rectangles in thisfigure which have horizontal bars within the rectangle. The introns arerepresented by the thin line connecting each of the exons. Therectangles having diagonal lines represent untranslated sequences. Thestart codon is represented by ATG and the stop codon is represented byTGA. The entire sequence ends with a poly-A tail.

EXAMPLE 18

This example identified single nucleotide polymorphisms (SNPs) of CD151.

Materials and Methods:

Each of the introns will be analyzed by PCR cloning to determine thedifferences in SNPs and to correlate these SNPs with the susceptibilityof cells to PRRSV infection. The sequence data from each of the intronsfrom the PAM cells is obtained as described in Example 17 and theprimers are designed to amplify just the each of the introns and thenare used to amplify the each of the corresponding introns in the varioussusceptible and non-susceptible cell lines. The data obtained will thenbe analyzed using the GCG program (University of Wisconsin, Madison,Wis.).

Results:

The single nucleotide polymorphism in the introns and exons of any genemay play important roles in gene expression. The sum of the sequencevariation of the introns may also play an important role in theexpression of the gene in the cell thereby leading to variations insusceptibility to PRRSV infection.

EXAMPLE 19

This example determines the chromosomal localization of porcine CD 151.

Materials and Methods:

Chromosomal localization is performed by Flourescent In-SituHybridization (FISH). The chromosome spreads are prepared by standardmethods. The leukocytes will be collected from heparinized blood, thered blood cells (RBC's) are lysed and the chromosome is spread on theslides. The slides are denatured in 70% formamide in 2×SSC at 70° C. Theslides are then incubated with biotin labeled probe (genomic CD 151).The post-hybridization washes will be performed by standard methods andthe binding detected by standard methods using flourescein labeledavidin as described by Roland M. Nadore in Flourescence In-SituHybridization Using Whole Chromosome Library Probes, Methods InMolecular Biology; edited by Lorette Javis (pages 371–377), theteachings of which are hereby incorporated by reference (Nadore, RolandM. 1999 Immunocytochemical Methods and Protocols (2^(nd) ed.), Methodsin Molecular Biology, pages 371–377).

Results:

In addition to identifying the chromosomal localization of the porcineCD 151, this example also helps to localize the other diseasesassociated with this chromosome. This would provide a distinct advanceover the prior art in that the CD 151 gene may be linked to otherdisease resistance genes or production traits, which effect the breedingstatus, such as meat carcass quality traits. Gene location will helppredict the collateral effects of selection for CD 151. Once otherdiseases associated with this chromosome have been correlated with CD151, strategies for breeding can be developed. Of course, it would bemost desirable to be able to select for PRRSV resistance and stillmaintain carcass quality.

EXAMPLE 20

This example provides a transcriptional profile of the porcine CD 151gene.

Materials and Methods:

Various susceptible and nonsusceptible tissues were collected from pigsand the tissue was ground such that the RNA could be extracted using theacid guanidinium and phenol chloroform method as described byChomczynski and Sacchi, Single-step method of RNA isolation by AcidQuadnidinium thiocyanate-phenol-choloroform extraction. 62 Anal.Biochem., 156–159.(1987), the teachings of which are hereby incorporatedby reference. The total RNA was run on 1% formaldehyde agarose gel andtransferred to the nylon membrane. The CD 151 transcripts was thendetected by Northern Blotting using radiolabeled CD 151 mRNA probefollowing standard protocols. Equal amounts of RNA were loaded into eachwell. The sizes of the transcripts were compared by using the knownradiolabeled RNA markers. The transcript amount was quantified using theGAPDH mRNA probe as the control. After post-hybridization washes andautoradiographs, the results were recorded.

Results:

This example identified two transcripts of CD 151. The size of thesetranscripts is not specifically known. However, the first transcript islarger/upper and the second transcript is lower/smaller In the firstexperiment, both the upper and lower transcripts were present insusceptible tissues (spleen, lung, kidney and heart). However,nonsusceptible tissues (muscle and liver) had only the upper transcript.Thus, the larger (upper) transcript alone is indicative of PRRSVsusceptibility whereas the smaller (lower) transcript alone is presentin nonsusceptible tissues.

EXAMPLE 21

This example demonstrates the RNA-binding activity of porcine CD 151.

Materials and Methods:

Porcine tissue (100 mg) was frozen in liquid nitrogen and ground in amicro-dismembranator for 30 seconds. The tissue pellet was resuspendedin 100 μl of 0.01M phosphate buffered saline (PBS) and 900 μl of RIPAlysis buffer (10 mM TrisHCl pH 8.0, 0.14 M NaCl₂, 1% Triton X-100, 1%Sodium Deoxycholate, 0.1% SDS, and 0.2/mL of Aprotinin), vortexed andincubated at 37° C. for 30 minutes. The tissue sample was thencentrifuged at 8,000 rpm for 10 minutes to remove the debris. Thesupernatant was collected and used for immunoprecipitation with anti-CD151 monoclonal antibody (PharMingen, San Diego, Calif., Catalog No.556056) Followed by northwestern assay using radiolabeled PRRSV 3′ UTRRNA, as described above.

Results:

Porcine CD 151 has RNA binding activity as shown in the northwesternblot of FIG. 11. This blot is from muscle and clearly demonstrates theRNA binding activity of porcine CD 151. This verifies that porcine CD151 functions similarly to simian CD 151 and is responsible for RNAentry and is therefore the susceptibility factor for PRRSV.

The correct size and also the amount of each transcript will be analyzedand correlated with disease susceptibility of each tissue to PRRSV.

EXAMPLE 22

This example determines the cytopathology and viral titers in non-simiancell lines transfected with porcine CD 151.

Materials and Methods:

BHK-21 cells were stably transfected with porcine CD 151 as described inExample 1. The construct of porcine CD 151 used for this experimentlacked nine amino acids of the open reading frame. This clone wastransfected in BHK-21 and selected for with G-418 for stabletransfectants. This clone was then tested for PRRSV susceptibility. ThePRRSV titers were then tested by titration experiments. The PRRSV wasinfected in serial dilutions (1:10, then 10-fold dilutions further down)for one hour at 37° C. The monolayer was then washed in PBS and thenreplaced with 1 ml of MEM. The 100 μl of supernatant was collected at 0,24, 36, 48, and 72 hours post-infection. After 72 hours, the cells werestained for PRRSV by direct fluorescent antibody test (FA) using SDOW-17nucleoprotein mAB. The virus collected at different time intervals wastitrated in the MARC cells.

Results:

As expected, no cytopathic effects were observed in BHK-21 cells.However, the stable transfection of MARC cells with CD 151 did lead toenhanced PRRSV production. This provides further verification that theporcine CD 151 functions similarly to the other previously identified CD151 sequences. However, the construct without the first nine amino acidsdid not function quite as well. Thus, porcine CD 151 can be similarlyused to produce PRRSV in non-simian cell lines.

EXAMPLE 23

This example determined and compared the amount of CD 151 expressed indifferent tissues.

Methods and Materials:

Various susceptible and non-susceptible tissues were collected from pigsand the tissue was ground such that the RNA could be extracted using theacid guanidinium and phenol chloroform method as described byChomczynski and Sacchi. The total RNA was then run on 1% formaldehydeagarose gel and transferred to the nylon membrane. The CD 151transcripts were detected by Northern blotting using radiolabeled CD 151mRNA as the probe and following standard protocols. An equal amount ofRNA was loaded into each well and then the known radiolabeled RNAmarkers were used to compare the sizes of the transcripts. Thetranscript amount was quantified using the GAPDH mRNA probe as thecontrol. The results were recorded after post hybridization washes andautoradiographs.

Results:

Northern blot assays of porcine tissues for CD 151 mRNAs of any genewill indicate the amount of protein expressed in the tissue. Thepresence of the correct size and the amount of the transcript are veryimportant in determining protein expression in the tissue. Twotranscripts were observed as discussed previously. Each tissue has itsown transcriptional profile for CD151.

EXAMPLE 24

This example compares the transcriptional profiles of tissues as shownby Example 20 and compares the results for different breeds of pigs.

Methods and Materials:

Transcriptional profiles are prepared as described in Example 20.However, differences in these profiles are correlated with varioussusceptible and non-susceptible breeds of pigs.

Results:

The amount of each transcript in different tissues from different breedsof pigs will be correlated with the relative PPRRSV susceptibility ofeach breed. Nonsusceptible breeds or breeds with reduced susceptibilityto PRRSV infection will show decreased levels of CD 151 in the tissueswhen compared with levels from these same tissues but from othersources. The transcriptional profile of individual pigs in differentbreeds of swine will be used to develop selection criteria for improvedswine breeding.

EXAMPLE 25

This example determines the tissue distribution of porcine CD 151.

Methods and Materials:

Different porcine tissues were collected an 100 mg of the tissue wasfrozen in liquid nitrogen and ground using a microdismembrator with apulse of 30 seconds. The ground tissues was resuspended in 1 ml of RIPAbuffer. The sample was then incubated at room temperature for 30 minutesin the shaker. The protein sample was then centrifuged at 6,000 rpm for5 minutes. The supernatant was then electrophorosed to a 10% SDS-PAGEgel. Next, the proteins were transferred to the nitrocellulose membraneblocked in 10% horse serum and the CD 151 antibody was added 1:500 inblocking solution. The membrane was then washed three times for 15minutes before incubating in secondary anti-mouse HRPO conjugate (VectorLaboratories, Burlingame, Calif.) (1:10,000). The membranes were thendeveloped in membrane TMB conjugate (Kirkegaard & Perry Laboratories,Inc., Gaithersburg, Md.).

In order to determine the genomic organization of porcine CD 151, ananti-CD 151 monoclonal antibody 14A2.H1 (Pharmingen, San Diego, Calif.)raised against human CD 151 was used. A western blot assay was thenperformed and the results analyzed for CD 151 expression levels. Theamount of two isoforms of porcine CD 151 will be quantified andcorrelated with PRRSV susceptibility. These parameters will also be usedto aid in the selection of swine for breeding. Ultimately, the criteriacould be assigned different weights for breeding and selection.

Results:

It was shown that pig muscle and heart tissue have large amounts of CD151 as two bands were identified using the western blot assay. One bandwas approximately 60 kDA and the other band was approximately 30 kDA.Because the expression of the CD 151 gene within a cell is determined bythe genomic organization of the gene, the expression of CD 151 in highquantities or in low quantities is directly related to thisorganization. The organization also indicates whether there are otherfactors such as enhancers or inhibitors of gene expression leading tothe different susceptibility of pigs to PRRS disease.

EXAMPLE 26

This example illustrates the critical role of the YRSL domain which ispresent at the carboxy end of CD 151 in the biology of PRRSV.

Materials and Methods

To study the effect of mutations of the cytoplasmic tails of CD 151 onPRRSV RNA entry, the effects of these mutations will be checked by theireffects on PRRSV infectivity. The presence of the virus will be checkedby immunofluorescence assay. Briefly, PCR-based mutogenesis will beperformed on two constructs of CD 151 (simian and porcine) to study theeffect of these mutations on PRRSV infectivity.

Results:

The CD 151 molecule contains a YRSL (SEQ ID No 29) sequence at thecytoplasmic tail. It is believed that this sequence is in the YXXL (SEQID No 36) motif or domain. This motif and especially the YRSL sequenceis a known RNA binding domain located at the carboxy terminal. In such adomain, the XX residues may be mutated without any change ininternalization. The L residue is a critical hydrophobic residue. If thecytoplasmic tail of CD 151 plays a critical role in PRRSV RNA entry, themutated CD 151 will be unable to restore the PRRSV infectivity in theBHK-21 cell line. It is believed that this will be the case if the Y orL are mutated. However, if the R or S are mutated, it is believed thatthere will be no effect on the RNA binding ability of the cytoplasmictail. Accordingly, such mutated CD 151 sequences would be able torestore PRRSV infectivity in the BHK-21 cell line. In such situations,it is believed that the YRSL sequence acts as one of the signals at thecarboxy terminal of CD 151. This would correlate with other known thingsthat enter cells at the end of the endocytic cycle which have signals attheir carboxy terminals. Endocytosis includes the formation of clathrincoated pits that bud from the cytoplasmic membrane into the cell. PRRSVenters through clathrin coated pits. It is known that CD 151 islocalized in endosomes of target organs such as endothelial cells andmay participate in the entry of PRRSV through endosomes. PRRS has manygenotypes and over 75 immunotypes but all isolates use a common entrypathway. Accordingly, the information developed from this example wouldbe used to find out if any lines of pigs have mutations in this criticalsequence of CD 151.

Discussion

Viruses are obligatory internal parasites that have limited geneticmaterial for replication. Viruses often utilize host cellular factors,in the form of RNA binding proteins, transcription factors, proteases,and membrane factors for their replication. RNA binding proteins couldplay roles in transcription, translation, orientation, and transport ofviral RNA. To understand how viral RNA replication proceeds, it isimportant to identify and characterize these proteins. There is noreport of identification of host cellular proteins binding to the 3′ UTRof PRRSV.

Here the identification of a novel 29 kD glycoprotein, simian CD 151,which binds to the 3′UTR of PRRSV is reported. CD 151 is a transmembraneglycoprotein, belonging to the tetraspan or transmembrane 4 superfamilyof cellular proteins. Tetraspans have four highly conserved hydrophobicdomains spanning the lipid bilayer and two extracellular domains. The Nand C terminals are found in the cytoplasm. The simian CD 151 shares 98%homology with PETA-3, which is found mainly in endothelial cellmembranes, platelets and megakaryocytes, epithelium, lung, muscle,Schwann cells, and glomeruli. This protein has been shown to beupregulated in human T cells transformed by T cell leukemia virus type 1and has been named SFA-1. Significant amounts of PETA-3 are present inintracellular compartments, localized to perinuclear vesicles,accounting for 66% of the total amount of PETA-3 protein present inendothelial cells. Tetraspan CD 151 is required for the early step inmetastasis of tumors and is involved in interactions with the integrinsin hemopoietic cell lines, modulation of cell-cell adhesions, andtransmembrane signaling through protein-protein interactions.

After demonstrating that CD 151 binds to the 3 ′UTR of PRRSV, thecorrelation between the presence of CD 151 and susceptibility to PRRSVinfection was tested. It was found that MA 104, MARC, COS-7 and Verocell lines which are all derived from kidney cells have CD 151. However,BHK-21 and MDBK cells are also derived from kidney cells, but they lackCD 151. Thus, CD 151 as a kidney specific protein is ruled out. ThePRRSV does not enter BHK-21 cells, but the transfection of these cellswith either the PRRSV RNA or the infectious cDNA clone results inproductive viral infection without spreading to neighboring cells. Thus,it was reasoned that although BHK-21 cells support the replication ofPRRSV RNA, they lack the cellular factors required for the entry of thevirus or viral RNA. Since it was found that CD 151 is a transmembraneprotein, it was reasoned that it might have a role in entry of PRRSVand/or viral RNA. There is a previous report that CD 9, anothertetraspan, renders MDBK a non-susceptible cell line, susceptible tocanine distemper virus infection and functions as an entry molecule. Toprove that CD 151 is one of the susceptibility factors of PRRSVinfection, it was determined that BHK-21 cells expressing CD 151 getinfected by PRRSV while the parent cell line lacking CD 151 did not getinfected. Thus, because CD 151 is a transmembrane molecule, it mightplay a role in virus entry or viral RNA entry into BHK-21 cells.

Entry of the virus into cells occurs by the interaction of viralproteins with specific viral receptors present on the cell membrane.These receptors are very important determinants of susceptibility toviral infection. Previous studies have demonstrated that PRRSV binds toa heparin-like molecule, and entry of the virus occurs byreceptor-mediated endocytosis. Low pH in these vesicles is essential forthe productive infection. Coimmunoprecipitation was performed todetermine if there is any interaction between PRRSV proteins and CD 151,however, there was no evidence for direct protein-protein interactionbetween them. This is in agreement to the previous observation that CD9, another tetraspan is a susceptibility factor for canine distemper butit does not directly interact with the viral proteins. For a protein tobe a receptor, it has to interact with the viral proteins; thus, the CD151 does not function as the receptor. Because CD 151 is present in thevesicles and also in the membranes, its role as an RNA transportermolecule is predicted based on two observations: first, it renderednon-susceptible BHK-21 cells susceptible to viral infection andsecondly, MARC cells that overexpress CD 151 (by stable transfection)exhibit a 100-fold increase in virus production as compared to thenative cell line. This effect of CD 151 on viral replication may beexplained by increased cellular uptake of the viral RNA during the entryof the virus with CD 151 functioning as an RNA-transport molecule. Thisprocess is shown schematically in FIG. 9. There are previous data wherethe RNA binding proteins are predicted to function as RNA transportermolecules. Testis/brain RNA binding protein interacts with the 3′UTR ofthe mRNA and it transports mRNA to specific intracellular sites wherethey are translated. This protein is also reported to transport themRNAs between cells. In tobacco mosaic virus, a 30 kD movement proteinis involved in transport of the viral RNA from cell to cell via theplasmodesmata. In beet necrotic yellow virus, proteins p 42, p 14, and p13 are involved in the movement of viral RNA through the plasmodesmataby interaction with the viral RNA. There is no report of an RNAtransport molecule in animal viruses. In PRRSV infection, the CD 151either alone or in conjugation with other proteins might act as the RNAtransporter molecule for entry into the cytoplasm after the virusuncoats in the vesicles. Thus, the present invention demonstrates thatCD 151 is involved in the replication of PRRS virus mainly as a viralRNA entry molecule.

To determine whether the CD 151 transmembrane protein plays a role inentry of the virus/viral RNA into the cell, BHK-21 cells were chosen fortesting because BHK-21 cells are resistant to PRRSV infection. However,when BHK-21 cells are transfected with either the viral RNA or theinfectious cDNA clone of PRRSV, they support the replication of thevirus. Thus the resistance factor in BHK-21 cells might be associatedwith the entry molecule. The fact that BHK-21 cells lack CD 151 wasdefinitively shown by RT-PCR (FIG. 5, lane 6) using CD 151 specificprimers. BHK-21 cells expressing CD 151 were obtained by stabletransfection as described above in the Materials and Methods. Thesetransfected cells were infected with PRRSV and tested for virusproduction by immunohistochemistry. Notably, it was found that the CD151 transfected cells became infected with PRRSV but the parent BHK-21cells lacking CD 151 (verified in FIG. 6) did not become infected.

Another advantageous aspect of the present invention is that the babyhamster kidney (BHK) cell line used in these experiments is not ofsimian origin. Thus it differs from all other cell lines used forpropagating PRRSV which are of simian origin. This provides a greatadvantage because the use of non-simian cells lines poses no risk oftransmission of primate or simian viruses to pigs, especiallyxenotransplanted pigs. By using non-simian cell lines to propagatePRRSV, primate and/or simian viruses will not be introduced into swinepopulations and will therefore not pose a risk to the human population.Also, because these cells expressing CD 151 have higher titers of PRRSV,they are more convenient and economically useful for propagating PRRSVfor vaccine production. Advantageously, these cells can be used to makekilled and live PRRS virus vaccines and serodiagnostic assays.

Finally, the present invention determined the DNA sequence and genomicorganization of porcine CD 151. Such knowledge will aid in thedevelopment of a DNA-based PCR which could be done on ear-notchedbiopsies. If a live animal was not available, testing could also be doneusing germplasm, semen, and flushed ova. Such a design permits germplasmfrom deceased animals to be used several years and generations afterdeath in order to improve the herd as a whole. If was further found thatporcine CD 151 had PRRSV RNA-binding activity in swine tissues and thatporcine CD 151 had only 84% sequence homology with known CD 151sequences for humans, monkeys, and mice. Additionally, the presentinvention has enabled the determination of porcine CD 151 mRNA. Such asequence can be determined by combining the exons of Table 2 insequential order and allowing for the transcription from the DNAsequences of these exons into RNA. Such a sequence which can lead tomRNA sequence is provided herein as SEQ ID No. 38.

A monoclonal antibody designated 7G10 has been developed. this antibodycompletely blocks PRRSV infection in cell culture. This antibody wasmade against a host cell protein of the MARC cell line and is an IgAisotype expressing high neutralizing activity against PRRSV. On thebasis of virus overlay protein blot assay (VOPA), 7G10 recognizes areceptor of PRRSV and is thus able to cause the neutralization of PRRSV.These specific characteristics make 7G10 applicable in the swineindustry for preventing the transmission of PRRSV.

1. An isolated plasmid containing the DNA sequence of SEQ ID No.
 14. 2.An isolated vector containing the DNA sequence of SEQ ID No.
 14. 3. Theisolated DNA sequence of SEQ ID No.
 14. 4. A transformed cell linecontaining the DNA sequence of SEQ ID No. 14.